Download 1 - BioMed Central

Supplementary Information
Supplementary Methods:
Identification of putative chicken-zebra finch orthologous alignments
As described in the Methods section, the zebra finch genome was compared with the
chicken genome. Chicken Refseq protein sequences (19,661) and zebra finch
expressed sequence tags and mRNAs (67,671) downloaded from GenBank
(www.ncbi.nlm.nih.gov) were cleaned of vector contaminants using SeqClean
(http://www.tigr.org/tdb/tgi/software/), and repetitive sequences were masked using
RepeatMasker (http://www.repeatmasker.org). The TIGR gene indices clustering
tools (Tgicl) [10] was used to cluster the zebra finch sequences with a minimum
length of 100 bases and identity of 96% for overlapping regions into 9,716 consensus
contigs. These zebra finch contigs were searched against the chicken protein
sequences using Blastx [11], with an E value ≤ e-10 separating best hits for each
protein from paralogous sequences. The best-hit protein pairs identified in the Blastx
search were aligned using T-Coffee [12]. Alignments of length < 70 amino acids and
sequence identity < 60% were discarded to remove short and spurious sequences.
These protein alignments were then used as templates to generate 3,653 pairwise
coding sequence (CDS) alignments that were used in subsequent analyses.
Resequencing:
Table S1. Sets of primer pair sequences and their associated optimal PCR parameters.
Amplicon
Size
(bp)
1
903
2
799
3
684
4
708
5
943
6
867
7
970
8
906
Orientation
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
TM
(oC)
[MgCl]
(mM)
56
15
60
20
56
20
61
15
57
25
58
20
60
20
62
20
Primer Sequences
GGTTAGGTTGCAAGGTTTTGTC
CCAGCCCTTAAGATTTCATGTC
GAATCCTAACATCCAGCAAAGC
AGTGAAGAACACACACCACCAC
CAGGAAAAATCCCAACTGAAAG
GCACTACTTGGCAAACACTCTG
CAGAGTGTTTGCCAAGTAGTGC
ACATACTGGTGCCATTGAACTG
ACAGTTCAATGGCACCAGTATG
TTCAGGCCTTCTCACTAAGCTC
GCAGTGCTTGTTGATGAATACC
TTAGATGCCAACTGTGTTGTCC
AATGCAGTTTTAACCCCTGAGA
GGGTTAAAGACGGTAACAAGCA
ACAATTGCAGTACAACCAGCAG
TCAAACACTCATGGCCATCTAC
Table S2. PCR cycle program used for each primer pair.
Step Temp. (oC)
1
95
2
95
3
TM
4
72
5
72
Duration
15 mins
30 seconds
45 seconds
60 seconds
15 mins
TM is the annealing temperature as listed in Table S1. Steps 2 to 4 were repeated 33
times in sequence.
PMut
In some cases, the program did not have sufficient confidence in the results due to the
high protein sequence divergence between chicken and other well-studied species
with which it was compared. Thus the prediction outcomes were also classed as not
determined in those cases.
IL-4Rα proximity to other immune genes
Situated on chromosome 14, the 5’ end of IL-4Rα is just 150 bp from a transcribed
element (NSMCE1) [18]. The IL-21 receptor is near the 3’ end of IL-4Rα and an IL9R precursor homolog lies close to the IL-21R as well.
Identification of IL-4Rα in the zebra finch
A tBlastn search [81] of the zebra finch genome sequence (July 2008 assembly)
against the chicken gene protein sequence (XP_414885) and the translated versions of
zebra finch sequences (DQ213788, DQ231787) identified the IL-4Rα gene on zebra
finch chromosome 14. Alignments of known bird IL-4Rα gene sequences and the
candidate region on chr14 using T-Coffee [12] and the tBlastn data yields a large
portion of the translated zebra finch IL-4Rα coding sequence.
The zebra finch IL-4Rα gene
The GenBank zebra finch IL-4Rα mRNAs include 5’ UTR and perhaps a leader
sequence, like the chicken copy. The zebra finch IL-4Rα coding region starts with
exon 1 at base chr14:16,260,749 is 69 bases long. Other identifiable orthologous
coding regions to chicken are exon 2 at 16,264,259-402, exon 3 at 16,265,272-427,
exon 4 at 16,266,443-604, exon 5 at 16,267,141-299, exon 8 at 16,268,959-9,036 and
exon 9 at 16,269,132-71,277. Regions for exons 6, 7 and 10 were not clear as the
zebra finch mRNA sequences are short and the divergence between chicken and zebra
finch is high at the 3’ end of the gene, which is reflected in the number of segregating
polymorphism in the chicken samples.
An alignment of IL-4Rα protein sequences from the zebra finch and other bird
sequences using T-Coffee [12] was examined for substitutions among species.
[81] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local
alignment search tool. J. Mol. Biol. 1990, 215:403-410.
2
Supplementary Results:
IL-4Rα Codeml Alignment Parameters
The IL-4Rα mRNA sequence (XM_414885) aligned as a best hit to 2 clustered zebra
finch mRNAs with a Blastx score of 339 and an e-value of 2e-92. A LRT of the
variable and fixed model pairwise comparisons showed that the variable model has a
log-likelihood of -1422.79 with ω = 0.5098. The neutral model log-likelihood with ω
= 1 was -1427.74, so the variable model was significantly more likely with p =
0.0017.
Pairwise comparisons of chicken and zebra finch genes:
A set of 3,653 chicken and zebra finch CDS pairwise alignments were examined to
identify candidate genes potentially subject to directional selection. Ten candidate
genes were observed with ω > 0.5 where the variable model was significantly
favoured. The most represented functional category among the 10 candidate genes
was related to immunity. Three genes have roles in the immune response: IL-4Rα,
protein inhibitor of activated STAT 2 (Pias2) and progesterone-induced blocking
factor 1 (Pibf). Other functional categories included apoptosis (G-2 and S-phase
expressed 1), signalling (a phosphatase and an anion exchanger), and intracellular
structure (NADH DH 1β 6 and GORASP2). Functions for two genes were unknown.
Two of the genes with ω > 1 were not valid coding sequences; the other two were a
phosphatase (PPM1K) and an unannotated sequence.
Chicken genes identified that interact with IL-4Rα:
Interestingly, the two other chicken immune genes identified by this pairwise
comparison method (Pibf and Pias2) have human orthologs that interact with each
another and IL-4Rα. Human Pibf is an immunoregulatory factor expressed during
embryo development that regulates TH1 and TH2 cytokine production balance by
binding the IL-4Rα and an anchored Pibf receptor chain, which activates Janus kinase
1 (Jak1) to phosphorylate STAT6 [60]. Normally, activated STAT6 proteins dimerise
and translocate to the nucleus, where they activate TH2 cytokines [61-62]. However,
human Pias proteins may prevent cytokine activation by inhibiting STAT proteins in
the nucleus [63-64]. Thus the 3 immune genes identified with this method not only
are expected to interact in the same pathway but also are likely to have crucial roles in
modulating the immune response of chickens to viruses, bacteria and parasites.
3
Table S3. Pairwise comparison details and functions for chicken sequences with ω >
0.5 and p < 0.05.
CK refseq
ZF Contig
ω=dN/dS
2ΔML
P value
dN
dS
Chicken
gene name
ZF contig
information
Human
Function
XM_420574
CL1281Contig1
3.0968
5.668
0.0173
3.7408
1.2080
LOC422614
Phosphatase
Signalling
XM_414705
CL522Contig1
0.5373
4.334
0.0374
0.2246
0.4180
NDUFB6
NADH DH
1β
Structure
XM_419473
CL560Contig1
0.5162
4.776
0.0289
0.2010
0.3895
SLC4A1AP
Kanadaptin
Signalling
NM_001012594
CL6285Contig1
0.5127
4.430
0.0353
0.1569
0.3061
GORASP2
-
Structure
NM_001031332
CL4994Contig1
0.5511
8.708
0.0032
0.2542
0.4612
GTSE1
Apoptosis
XM_414885 *
CL6154Contig1
0.5098
9.896
0.0017
0.1791
0.3514
LOC416585
IL-4R αchain
NM_001030626
CL4084Contig1
0.5665
6.796
0.0091
0.4255
0.7511
PIAS2
-
Immunity
XM_417014
CL4220Contig1
0.5666
4.006
0.0453
0.1145
0.2021
LOC418820
-
Immunity
XM_420836
CL4938Contig1
CL3015Contig1
CL2548Contig1
4.0730
0.5215
0.5082
6.172
3.988
7.616
0.0130
0.0458
0.0058
4.0265
0.1965
0.5438
0.9886
0.3768
1.0701
LOC422894
Gal-4NAc4-sulfotr
-
Protein phosphatase 1K (PP2C
domain containing)
NADH dehydrogenase
(ubiquinone) 1 beta
subcomplex, 6, 17kDa
Solute carrier family 4 (anion
exchanger), member 1,
adaptor
Golgi reassembly stacking
protein 2, 55kDa
G-2 and S-phase expressed 1
Interleukin-4 receptor alphachain
Protein inhibitor of activated
STAT, 2
Progesterone-induced
blocking factor 1
-
-
-
XM_001234647
LOC771361
Immunity
Chicken GenBank description
* IL-4Rα, the chicken gene selected for resequencing. 2ΔML is twice the difference of
the maximum likelihood values of the variable model minus the fixed model.
Table S4. Estimated distribution of synonymous (S.dS) and nonsynonymous (N.dN)
SNPs by the codeml free-ratio model.
Sample
Chicken 1
Red JF 1
Grey JF 1
Ceylon JF 1
Green JF 1
Bamboo partridge
Grey francolin
N.dN
5.9
2.0
1.0
3.0
32.2
20.0
22.7
S.dS
5.5
4.4
0
0
16.8
12.9
8.7
1
On the branch ancestral to the Gallus birds, 19.8 nonsynonymous and 6.8
synonymous mutations were observed.
4
Additional file 1. An alignment of chicken and human IL-4Rα protein sequences.
The consensus human IL-4Rα sequence isoform a (GenBank accession number
NP_000409) and the consensus chicken sequence (XP_414885) were aligned with TCoffee [12]. The sites marked green were subsequently found to be candidates for
selection according to PAML M8 BEB results. Sites marked green and in red letters
indicate those subsequently observed as segregating in chicken populations and/or
with differences between the chicken and the red JF sequences.
5
Additional file 3. The numbers of genes (N) in classes of ω values from pairwise
alignments of chicken-zebra finch gene sets where the variable model was favoured
(p<0.05).
1000
100
N
10
1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
ω
The y-axis is on a logarithmic scale. The ω values on the x-axis are classes into
groups of 0.01, with the exception of values greater than 1, which are classed as 0.991.00.
Additional file 4. Codeml neighbour-joining phylogeny of IL-4Rα.
Branch lengths were estimated by
maximum likelihood under the free-ratio
model, which assumes an independent ωratio for each branch: these values are
displayed. The branch length displayed is
0.1 of the total branch lengths for the tree.
The ω for chicken was 0.4181 when
sample FJ542675 was used instead of
FJ542575. The ω values for grey and
Ceylon JF are high because no
synonymous SNPs were observed.
6
Table S5. Protein function impacts predicted by PMut for candidate M8 BEB sites
under selection and polymorphic between the chicken and the red JF genome
sequence.
Base
Position
4435-37
12652-54
12742-44
12871-73
12985-87
13096-98
13096-98
13096-98
Amino Acid
Position Red JF
5
F
517
Q
547 1
L
590
S
628 2
D
665
M
665
M
665 1
R
Alt.
L
H3
I
G
E
R
Q4
Q4
Prediction
Score
Certainty
Outcome
neutral
neutral
neutral
neutral
neutral
neutral
neutral
neutral
0.316
0.095
0.026
0.329
0.035
0.495
0.119
0.510
3
8
9
3
9
0
7
5
not significant
neutral
neutral
not significant
neutral
not significant
neutral
not significant
Alt. stands for alternative allele. 1 Polymorphic between the chicken and the red JF
sample. 2 The red JF allele was the same for the genome sequence and sample. 3 The
red JF sample and some chickens shared a synonymous SNP at this site. 4 The chicken
minor allele at the site. Substitutions where the PMut certainty values ≤ 6 did not have
statistical support for the predicted change.
7
Additional file 5. Genotypes at SNP sites polymorphic in the chicken for all samples.
The coding sites are marked as “Y” if nonsynonymous. Samples are from Pakistan
(FJ542565-FJ542584), Burkina Faso (FJ542585-FJ542604), Senegal (FJ542605FJ542624), Sri Lanka (FJ542625-FJ542644), Botswana (FJ542645-FJ542664),
Bangladesh (FJ542665-FJ542684), Kenya (FJ542685-FJ542704), Broilers
(FJ542705-FJ542744), bamboo partridge (FJ542745-6), grey francolin (FJ542747-8),
green JF (FJ542749-50), grey JF (FJ542751-2), Ceylon JF (FJ542753-4) and red JF
(FJ542755-6). Bases with nucleotide A are in green, C in blue, G in yellow and T in
red.
8
Table S6. Tajima’s D and Fay and Wu’s H for each Asian and African population.
For each population, 10 chickens were sampled each.
Continent
Asia
Africa
Population
Bangladesh
Pakistan
Sri Lanka
Botswana
SNPs
Tajima’s D
P value
Fay & Wu’s H
P value
82
0.85
0.032
-14.33
0.031
86
1.10
0.006
-17.58
0.027
73
0.86
0.033
-22.23
0.004
74
0.90
0.015
-22.32
0.003
Burkina
Faso
51
1.21
0.004
-21.85
<0.001
Senegal
Kenya
70
1.03
0.012
-14.22
0.016
71
1.81
<0.001
-12.41
0.037
Additional file 6. Median-joining networks of haplotypes for all SNPs classed
according to the major groups at amino acids 5 (F5L) and 520 (L520P) from Figure
2b.
The four possible genotypes at these positions are denoted in the legend. Branch
lengths are proportional to the number of mutational differences between haplotypes.
The outgroup sample branch lengths are considerably reduced in order to show the
details of the chicken population network. V represents the green JF sequences; F the
grey francolin; B the bamboo partridge; G the grey JF; C the Ceylon JF; R the red JF
sample genotypes; and RJF the genome sequence.
9
Additional file 8. A multiple sequence alignment of zebra finch and other bird
samples protein-coding sequences.
Sites marked were candidates for selection according to PAML M8 BEB results (red),
and had differences in the chicken populations compared to the red JF genome or
samples (green). Regions marked with X were not resequenced. Bamboo refers to the
bamboo partridge. Chicken has 2 alleles (F, L) at site 5; red JF, grey JF and bamboo
partridge all have F; and Ceylon JF, green JF and grey francolin have L. At site 520
the alleles segregating in chicken (L, P) were present in chicken and red JF, and
though zebra finch genome has L, the remaining birds all had P.
10